Recombinant Expression Vector for Secretion of IL-21 and Attenuated Salmonella Transformed Therewith

Abstract

The present invention relates to a recombinant expression vector for the secretion of interleukin-21 and an attenuated Salmonella strain transformed therefrom, wherein the recombinant expression vector comprising a flgM gene and an interleukin-21 gene and the attenuated Salmonella strain transformed therefrom and the present invention provides a pharmaceutical composition for treating cancer comprising said attenuated Salmonella strain as an active ingredient.

Description

Technology Field

[0001] This invention relates to a recombinant expression vector for the secretion of interleukin-21 and an attenuated Salmonella strain transformed therefrom. This patent application claims priority to Korean Patent Application No. 10-2022-0092017 filed with the Korean Intellectual Property Office on July 25, 2022, the disclosures of said patent application are incorporated herein by reference. Background Technology

[0002] Cancer is a life-threatening disease in which the proliferative activity of cells does not stop, invading surrounding tissues and destroying normal cells. As the body's regulatory capacity declines due to aging, it becomes more vulnerable to cancer; in an aging society, cancer holds the undisputed number one spot among all causes of death, exceeding twice the mortality rate of heart disease, which ranks second.

[0003] While the most effective response system for treating disease is to strengthen immunity, cancer cells evade the body's immune response because they are not external invaders. Certain viruses or bacteria infect cancer cells more frequently than normal cells, and infected bacteria can become targets of attack by immune cells. Accordingly, anticancer treatment methods have been proposed that involve intentionally infecting the body with specific viruses or bacteria to stimulate the immune response and enable it to fight against cancer cells. Infectious bacteria such as Shigella, Vibrioholera, and pathogenic E. coli only penetrate the cells of the intestinal tract and do not reach the liver and spleen, which are vital organs for triggering immune responses. In contrast, Salmonella can penetrate the spleen and liver through the lymph nodes and stimulate a systemic immune response. Specifically, Leschner et al. (J. Mol. Med. 2010, 88,763-773) infected mice with CT26 tumors with fluorescent Salmonella via intravenous injection and tracked the infection pathways over time. The results showed that immediately after infection, the entire body was infected through the mice's blood; 20 minutes after infection, Salmonella accumulated in the spleen and liver; and 24 hours later, Salmonella was found to be concentrated only in the tumor tissue.

[0004] However, Salmonella is a representative bacterium that causes food poisoning and can be life-threatening by inducing sepsis through infection, making it too pathogenic to be used directly for cancer treatment. A research team at Yale University announced that genetically modifying Salmonella can weaken it by removing only its toxicity while retaining its tumor-attacking characteristics, and that injecting this weakened Salmonella can suppress tumors by inducing immune stimulation. However, weakened Salmonella has low viability and reduced immune-inducing ability, and there is a concern that mutations could cause the weakened strain to revert to wild-type Salmonella, leading to sepsis. Furthermore, due to a lack of research and development funding, anticancer treatments using weakened Salmonella have mostly been halted or suspended at the first stage of clinical trials.

[0005] The induction of sepsis that may accompany cancer treatment using bacteria such as Salmonella is a critical issue that must be overcome, and furthermore, the stimulation of the immune response must also be actively facilitated. Accordingly, research primarily focuses on developing bacterial strains capable of additionally expressing or suppressing substances beneficial for anticancer treatment, along with attenuation and immune activation, and utilizing these strains for anticancer therapy. Registered Patent No. 10-0852687 discloses a Salmonella strain expressing Tumor Necrosis Factor Alpha by transfecting a Tumor Necrosis Factor Alpha protein vector into an attenuated Salmonella strain, and an anticancer therapeutic composition containing the same; meanwhile, Published Patent No. 10-2021-0123556 discloses an anticancer Salmonella that induces an immune response through filament growth regulation.

[0006] “Interleukin-21” is extensively involved in the differentiation and function of lymphocytes and bone marrow cells and regulates both innate and adaptive immunity. In particular, in cancer treatment, IL-21 is known to have potential as an anticancer agent by enhancing the ability of CD8+ T cells and NK cells to kill tumor cells.

[0007] Accordingly, the inventors prepared a recombinant expression vector comprising the flgM gene; the interleukin-21 gene; and the flhDC gene, and an attenuated Salmonella strain transformed with said recombinant expression vector, and confirmed the excellent cancer treatment effect thereof, thereby completing the present invention.

[0008] The information described above in the background section is intended solely to enhance understanding of the background of the present invention and may not include information that forms prior art already known to those skilled in the art to which the present invention belongs. The problem to be solved

[0009] One aspect provides a recombinant expression vector comprising a flgM gene; an interleukin-21 gene; and a flhDC gene, wherein the flgM gene; the interleukin-21 gene; and the flhDC gene are operably linked to an inducible promoter.

[0010] Another aspect provides an attenuated Salmonella strain transformed with the above-mentioned recombinant expression vector.

[0011] Another aspect provides a pharmaceutical composition for cancer treatment comprising the above-mentioned attenuated Salmonella strain as an active ingredient. means of solving the problem

[0012] One aspect may provide a recombinant expression vector comprising a flgM gene; an interleukin-21 gene; and a flhDC gene, wherein the flgM gene; the interleukin-21 gene; and the flhDC gene are operably linked to an inducible promoter.

[0013] The present invention relates to a recombinant expression vector comprising a gene encoding a fusion protein of flgM and interleukin-21, wherein the flgM gene and the interleukin-21 gene are linked directly or through a linker. Hereinafter, the fusion protein of flgM and interleukin-21 is referred to as “flgM-IL-21”.

[0014] In this specification, the term "flgM" refers to a protein (or the gene encoding such a protein) that inhibits factor σ, which is responsible for mobilizing RNA polymerases required for late flagellar transcription in the bacterial type III secretory system. The "type III secretory system" refers to a pathogenic substance delivery system developed in Gram-negative bacteria. It is a multi-protein complex structure acting as a syringe-like mechanism (Mota LJ et al, Ann Med. (2005); 37(4): 234-249) that injects pathogenically active proteins directly into the cytoplasm by passing through the host cell membrane. In the type III secretory system, while the hook-basal body—the basic structure capable of forming flagella on the cell membrane—is being formed, the flgM protein remains in the cytoplasm and acts as an anti-σ that interferes with the transcription of class III genes, such as the flagellin subunit FliC or the stator protein MotAB. 28 It acts as a factor. Subsequently, after the completion of the Hooke-basal body structure, a simultaneous change in substrate specificity within the flagellar secretion system occurs; as a result, flgM is secreted out of the cell through the central channel of the Hooke, and subsequently, within the cell, σ 28 --dependent transcription is initiated. The secretion mechanism of flgM through the process of flagellar formation in the type 3 secretory system is shown in Figure 2.

[0015] In this specification, the term "Interleukin-21 (IL-21)" refers to a cytokine that has a potent regulatory effect on the immune system, including natural killer (NK) cells and cytotoxic T cells, capable of destroying virus-infected cells or cancer cells. Clinical trials are currently underway for Interleukin-21 as a monotherapy for the treatment of melanoma, renal cell carcinoma, and metastatic colorectal cancer, and it is being studied as an anticancer agent in immunotherapy, such as when administered in combination with agents like cetuximab, an antibody targeting the epidermal growth factor receptor (EGFR). The sequence of Interleukin-21 can be obtained from the known database NCBI GeneBank.

[0016] According to one embodiment, the present invention enables the secretion of flgM through the strain's type 3 secretion system and the extracellular secretion of interleukin-21 along with the secretion of flgM through the strain's type 3 secretion system.

[0017] In this specification, the term “flhDC” refers to a primary regulator of the flagellin operon gene that can regulate the expression of the flgM gene. According to one embodiment, including the flhDC gene in the recombinant expression vector may contribute to enhancing the expression or secretion amount of flgM-IL-21.

[0018] In this specification, the term "gene" shall be considered in the broadest sense and may encode a structural protein or a regulatory protein. In this case, the regulatory protein may include transcription factors, heat shock proteins, or proteins involved in DNA / RNA replication, transcription, and / or translation.

[0019] In this specification, the term “genetic structure” refers to an assembly, functional unit, or construct containing genetic information of a target protein, and may be interpreted in the broadest sense. For the purposes of the present invention, the genetic structure is intended to enhance the secretion of flgM-IL-21 fusion protein from attenuated Salmonella strains and may refer, for example, to a DNA insert in which a flgM gene, a linker gene, and an interleukin-21 gene are operably linked.

[0020] In this specification, the term “vector” means a DNA product containing a base sequence encoding a target polynucleotide that is operably linked to a suitable regulatory sequence to enable the expression of the target polynucleotide within a suitable host. The regulatory sequence may include a promoter capable of initiating transcription, any operator sequence for regulating such transcription, a sequence coding for a suitable mRNA ribosome binding site, and a sequence regulating the termination of transcription and translation. After being transformed into a suitable host, the vector may replicate or function independently of the host genome and may be incorporated into the genome itself. The vector may be any that can replicate within a host cell. For example, the vector may be a plasmid, cosmid, virus, or bacteriophage in its natural or recombinant state. The vector may include a selection marker for selecting a host cell containing the vector. Selection markers are used to select cells transformed with a vector, and markers conferring selectable phenotypes such as drug resistance, nutritional requirements, resistance to cytotoxic agents, or expression of surface proteins may be used. For example, the marker may be one or more selection markers selected from the group consisting of ampicillin, neomycin, puromycin, hygromycin, and zeocin. The vector may include genes involved in replication and / or copy number regulation, such as a replication origin and a promoter, and may include, but is not limited to, restriction enzyme sites.

[0021] In this specification, the term “polynucleotide” may be in the form of RNA or DNA, said DNA including cDNA and synthetic DNA. The DNA may be single-stranded or double-stranded. If it is single-stranded, it may be a coding strand or a non-coding (antisense) strand, and said coding sequence may encode the same polypeptide as a result of degeneracy or redundancy of the genetic code.

[0022] The above polynucleotide may also include variants of the polynucleotide described herein, and the variants of the polynucleotide may be naturally occurring allelic variants of the polynucleotide or non-naturally occurring variants of the polynucleotide. An allelic variant is an alternate form of a polybase sequence that may have substitutions, deletions, or additions of one or more nucleotides that do not substantially alter the function of the polynucleotide being encoded. It is well known in the art that a single amino acid may be encoded by one or more nucleotide codons and that the polynucleotide can be easily modified to produce alternate polynucleotides encoding the same peptide.

[0023] According to one embodiment, the recombinant expression vector may contribute to enhancing the expression or secretion amount of flgM-IL-21 by including all of the flgM gene; the interleukin-21 gene; and the flhDC gene. For example, the recombinant expression vector may have enhanced expression or secretion ability of flgM-IL-21 by additionally including the flhDC gene in addition to the flgM gene and the interleukin-21 gene.

[0024] In this specification, the term "inducible promoter" refers to a promoter capable of switching the operation of a gene linked by an inducer, exemplified by the ara promoter, tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, T7 promoter, pBAD promoter, Tet promoter, trc promoter, pepT promoter, sulA promoter, pol 11(dinA) promoter, ruv promoter, uvrA promoter, uvrB promoter, uvrD promoter, umuDC promoter, lexA promoter, cea promoter, caa promoter, recN promoter, pagC promoter, hip promoter, and ansB It can be a promoter or a pflE promoter.

[0025] In this specification, "operably connected" may indicate that nucleotide sequences are connected on a single nucleic acid fragment so that one function is influenced by another.

[0026] In one embodiment, the flgM gene; the interleukin-21 gene; and the flhDC gene may have their expression regulated by the same inducible promoter. In one embodiment, the promoter may be an ara promoter.

[0027] According to one embodiment, the recombinant vector may have enhanced expression or secretion ability of flgM-IL-21 as the expression of the flgM gene; the interleukin-21 gene; and the flhDC gene is regulated by the same inducible promoter.

[0028] In one embodiment, the recombinant expression vector may include a flgM gene consisting of the polynucleotide sequence of SEQ ID NO. 1, an interleukin-21 gene consisting of the polynucleotide sequence of SEQ ID NO. 2, and a flhDC gene consisting of the polynucleotide sequence of SEQ ID NO. 3.

[0029] In one embodiment, the flgM gene and the interleukin-21 gene can be connected through a linker.

[0030] In this specification, the term "linker" refers to a polypeptide chain used to link a heterogeneous domain to a protein of interest or a gene encoding the same. The linker peptide may have a length of 2 to 50 amino acids, for example, 2 to 40, 2 to 30, 2 to 20, 2 to 15, 2 to 10, 2 to 5, 5 to 50, 5 to 40, 5 to 30, 5 to 20, 5 to 15, or 5 to 10 amino acids, but is not limited thereto. The linker peptide may be, for example, (G l S m ) n (l, m and n are each ≥ 2), (G l S m ) n- H p -(G l S m ) n (l, m and n are each ≥ 1, H ≥ 6), (G4S) a (EAAAK) b (G4S) a (a, b are integers from 1 to 4), (G4S) p (EAAAK) q (p, q are integers from 1 to 4), (EAAAK) x (G4S) y (x, y are integers from 1 to 4), A(EAAAK)4ALEA(EAAAK)4A, (GSSGGS) i(i is an integer from 1 to 4), KESGSVSSEQLAQFRSLD, EGKSSGSGSESKST, GSAGSAAGSGEF, (EAAAK) k (k is an integer from 1 to 5), may be any one selected from the group consisting of CRRRRRREAEAC, GGGGGGGG, GGGGGG, AEAAAKEAAAAKA, PAPAP, VSQTSKLTRAETVFPDV, PLGLWA, TRHRQPRGWE, AGNRVRRSVG, RRRRRRRR, GGSSHHHHHHSSGG, GGSSHHHHHHHHSSGG, and GSSGGSGSSGGSGGGDEADGSRGSQKAGVDE, but is not limited thereto. In one embodiment, the linker peptide may be GGSSHHHHHHHHSSGG.

[0031] A recombinant expression vector according to one embodiment may have enhanced expression or secretion ability of flgM-IL-21 by linking the flgM gene and the interleukin-21 gene through a gene encoding the GGSSHHHHHHHHSSGG linker peptide (SEQ ID NO. 4).

[0032] Another aspect may provide an attenuated Salmonella strain transformed with the above-mentioned recombinant expression vector. In the above-mentioned attenuated Salmonella strain, the terms and elements mentioned that are identical to those already mentioned are as described above.

[0033] In this specification, the term "transformation" means introducing a vector containing a polynucleotide encoding a target protein into a host cell so that the protein encoded by said polynucleotide can be expressed within the host cell. For example, said polynucleotide may be introduced into the host cell in the form of an expression cassette, or it may be introduced into the host cell in its own form and operably linked to a sequence required for expression in the host cell, but is not limited thereto.

[0034] In this specification, the term "attenuation" refers to artificially weakening the toxicity of a living pathogen, meaning that genes involved in the pathogen's essential metabolism are mutated so that the pathogen cannot cause disease within the body but stimulates only the immune system to induce immunity. Genes that cause attenuation of Salmonella are well known in the art, for example, the attenuated Salmonella strain may have lost the function of one or more genes selected from the group consisting of aroA, aroC, aroD, aroE, asd, Rpur, htrA, ompR, ompF, ompC, galE, cya, crp, cyp, phoP, phoQ, rfaY, dksA, hupA, sipC, clpB, clpP, clpX, pab, nadA, pncB, pmi, rpsL, hemA, rcsB, rfc, poxA, galU, cdt, pur, ssa, guaA, guaB, fliD, flgK, flgL, relA, and spoT. In order to prevent reversion to the wild type and further weaken toxicity upon in vivo application, the function of two or more of the above genes may be lost. The above-mentioned attenuated Salmonella strain possesses anticancer activity by targeting the tumor site and inducing an immune response.

[0035] It was confirmed that the expression and secretion levels of flgM-IL-21 were significantly enhanced when the attenuated Salmonella strain according to one embodiment was transformed with the recombinant expression vector, and through this, it was confirmed that an excellent anticancer effect was exhibited when a preparation containing the Salmonella strain was administered to an individual.

[0036] In one embodiment, the attenuated Salmonella strain may be a Salmonella strain in which the asd, rcsB, and galE genes are lost. More specifically, the attenuated Salmonella strain may be a Salmonella strain in which the functions of the aroA and aroD genes are additionally lost, so that the aroA, aroD, asd, rcsB, and galE genes are lost.

[0037] In one embodiment, the attenuated Salmonella strain may have an attenuation gene whose expression is regulated by a second inducible promoter. That is, the loss of function of a specific gene may be regulated by a second inducible promoter. Specifically, the attenuated Salmonella strain may additionally include a second inducible promoter in front of the attenuation gene to switch the attenuation. For example, when the attenuated Salmonella strain is injected into an individual in an attenuated state and targeting to a tumor site is completed, the strain is reverted to a wild-type Salmonella by the operation of the second inducible promoter, thereby inducing high immune activity compared to the attenuated Salmonella with low immune activity, which can lead to more effective cancer treatment.

[0038] In one embodiment, the attenuated Salmonella strain may have lost the galE gene, and the loss of function of the galE gene may be switched by the tetRA promoter. Accordingly, the attenuated Salmonella strain may be injected into an individual in an attenuated state to target a tumor site, and then the expression of the galE gene may be induced by doxycycline to induce high immune activity of the Salmonella strain. In this specification, the regulation of the loss of function of the galE gene is expressed as galE:tetRA.

[0039] In the case of the attenuated Salmonella strain according to one embodiment, the functions of the asd, rcsB, and galE genes may be lost, and it was confirmed that this attenuated Salmonella strain exhibits excellent flgM-IL-21 expression or secretion ability.

[0040] In one embodiment, the attenuated Salmonella strain may have enhanced secretion ability for the interleukin-21 protein. The attenuated Salmonella strain according to one embodiment is capable of not only expressing a peptide component having anticancer or cancer cell toxicity, but also enabling the secretion of said expressed peptide component in a target region, thereby exhibiting efficacy as a targeted anticancer therapeutic agent.

[0041] According to one embodiment, when an attenuated Salmonella strain is transformed with the recombinant expression vector, the expression of interleukin-21 is regulated in the presence of arabinose, and the interleukin-21 protein can be effectively secreted outside the strain by using flgM of the type 3 secretion system. Additionally, flhDC can also be regulated by the same inducible promoter to increase the expression amount of interleukin-21 secreted outside the cell.

[0042] In one embodiment, the attenuated Salmonella strain may be the attenuated Salmonella strain received under accession number KCTC15503BP.

[0043] Another aspect provides a pharmaceutical composition for anticancer purposes or a pharmaceutical composition for the prevention or treatment of cancer, comprising the above-mentioned attenuated Salmonella strain as an active ingredient.

[0044] In the above pharmaceutical composition, the terms and elements mentioned that are identical to those already mentioned are as described above.

[0045] The term "cancer," which is the target disease for prevention or treatment by the composition of the present invention, refers collectively to a disease caused by cells having aggressive characteristics in which cells divide and grow disregarding normal growth limits, invasive characteristics in which they infiltrate surrounding tissues, and metastatic characteristics in which they spread to other parts of the body. The cancer may be, for example, a solid tumor, and preferably, any tumor containing cancer tissue or cancer cells in which the cancer-targeting ability of the Salmonella strain can be exercised may be applied without limitation. For example, the types of cancer may be any one selected from the group consisting of pancreatic cancer, colorectal cancer, colon cancer, gastric cancer, liver cancer, colorectal cancer, brain cancer, breast cancer, thyroid cancer, bladder cancer, esophageal cancer, head and neck cancer, skin cancer, uterine cancer, and lung cancer, but are not limited thereto.

[0046] The composition of the present invention may be prepared by a method known in the pharmaceutical field for use as a drug for the prevention or treatment of cancer, and may be used either on its own or mixed with pharmaceutically acceptable carriers, forming agents, diluents, etc.

[0047] In this specification, "pharmaceutically acceptable carriers" may be determined in part by the specific composition being administered, as well as in part by the specific method used to administer the composition. Accordingly, suitable formulations of the composition are highly diverse, and, for example, the composition may be formulated for parenteral (i.e., intramuscular, intradermal, or subcutaneous) administration or nasopharyngeal (i.e., intranasal) administration. Generally, formulations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers may include water, alcohol / aqueous solutions, emulsions, or suspensions, as well as saline solution and buffered medium. The parenteral vehicle may include a sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or a fixative oil. Preservatives and other additives may also be present, for example, as antimicrobial agents, antioxidants, chelating agents, and inert gases.

[0048] The dosage of the active ingredient according to the present invention can be appropriately selected according to the absorption rate of the active ingredient in the body, the form of the preparation, the age, gender and condition of the patient, the severity of symptoms, etc., and the administration may be done once a day or divided into several doses. The general dosage is 0.001 mg / kg·day to 10 g / kg·day.

[0049] Another aspect provides a method for preventing or treating cancer, comprising the step of administering to an individual an amount of an anticancer pharmaceutical composition containing the attenuated Salmonella strain as an active ingredient that is effective for preventing or treating cancer.

[0050] In the method for preventing or treating the above cancer, the terms and elements mentioned that are identical to those already mentioned are as described above.

[0051] The above individual may be a mammal. The above mammal may be a human, mouse, rat, horse, dog, cat, cow, goat, or pig.

[0052] The above administration may be performed via any general route as long as it can reach the target tissue. For example, it may be administered via routes such as ophthalmic administration, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, transdermal patch administration, oral administration, nasal administration, pulmonary administration, and rectal administration, and specifically, it may be administered via routes such as ophthalmic administration depending on the intended purpose.

[0053] In this specification, the terms “treatment,” “treating,” “alleviating,” or “improving” are used interchangeably. These terms refer to a method of obtaining a favorable or desired result, including but not limited to therapeutic benefits and / or preventive benefits. A therapeutic benefit means any therapeutically significant improvement or effect thereon of one or more diseases, conditions, or symptoms under treatment. Regarding preventive benefits, the composition may be administered to a subject at risk of developing a specific disease, condition, or symptom, or to a subject reporting one or more physiological symptoms of a disease, even if the disease, condition, or symptom has not yet appeared.

[0054] In this specification, the terms “effective dose” or “therapeutic effective dose” refer to an amount of agent sufficient to produce a favorable or desired result. The therapeutic effective dose may vary depending on one or more of the subject and condition being treated, the subject’s body weight and age, the severity of the condition, and the method of administration, which can be readily determined by a person skilled in the art. Additionally, the terms apply to a dose that provides an image for detection by any of the imaging methods described herein. A specific dose may vary depending on one or more of the selected specific agent, the subsequent administration regimen, whether it is administered in combination with other compounds, the timing of administration, the tissue being imaged, and the body delivery system carrying it. Effects of the invention

[0055] According to a recombinant expression vector according to one aspect, it was confirmed that the expression and secretion of flgM-IL-21 were significantly enhanced in attenuated Salmonella strains transformed with the said recombinant expression vector.

[0056] In addition, according to a recombinant expression vector according to one aspect, it was confirmed that when a preparation containing an attenuated Salmonella strain transformed with the said recombinant expression vector was administered, it exhibited excellent anticancer effects and stability.

[0057] Therefore, a recombinant expression vector according to one aspect, and an attenuated Salmonella strain transformed therefrom can be utilized as an active ingredient of an anticancer composition. Brief explanation of the drawing

[0058] Figure 1 is a schematic diagram illustrating the extracellular secretion process of flgM-IL-21 using the type 3 secretion system. Figure 2 is a diagram briefly illustrating the flgM secretion mechanism using the flagellar formation process in the type 3 secretory system. Figure 3 is a diagram showing the cleavage map of the flgM-linker-IL-21-flhDC-pBAD18-asd+ plasmid according to one embodiment. Figure 4 shows the results of confirming the protein secretion levels of attenuated Salmonella strains transformed using flgM-linker1-IL-21-flhDC-pBAD18 and flgM-linker2-IL-21-flhDC-pBAD18 plasmids according to one embodiment. Lanes 1 and 2 show the results of confirming the flgM-IL-21 protein secretion levels in the whole cell culture medium, lanes 3 and 4 show the pellet, and lanes 5 and 6 show the supernatant. Figure 5 is a schematic diagram showing the processing of a tumor mouse model of a transformed attenuated Salmonella strain according to one embodiment. FIG. 6 is a graph showing the change in tumor volume in mice when mice are treated with a transformed attenuated Salmonella strain according to one embodiment (PBS: control group, uninduction: arabinose-untreated group, induction: arabinose-treated group). FIG. 7 is a graph showing the change in body weight of mice when treated with a transformed attenuated Salmonella strain according to one embodiment (PBS: control group, uninduction: arabinose untreated group, induction: arabinose treated group). Specific details for implementing the invention

[0059] The following examples will be explained in more detail. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

[0060] Example 1: Preparation of a plasmid for interleukin-21 secretion

[0061] 1-1. Preparation of insert DNA

[0062] To prepare a plasmid for interleukin-21 secretion, a genetic construct was prepared in which the flgM gene and the interleukin-21 gene were linked.

[0063] Specifically, a genetic construct according to one embodiment was constructed by linking the flgM DNA sequence of wild-type Salmonella (flgM anti-sigma-28 factor FlgM [Salmonella enterica subsp. enterica serovar Typhimurium str. LT2] Gene ID: 1252690, Locus tagSTM1172 NC_003197.2 (1257036..1257341, complement)) with the DNA sequence obtained through codon optimization for IL-21 (Salmonella). For codon optimization of IL-21, 130 amino acids of IL-21 were replaced with DNA, and TAA was inserted at the end.

[0064] The above flgM DNA sequence and IL-21 DNA sequence were linked using a linker, and to increase the expression of the flgM gene, 15 Shine-Dalgarno sequences (AGGAGGTTTGATCCT) were inserted in front of the flgM gene. Additionally, for cloning, a Nhe I site was inserted at the beginning, and a Sac I site was inserted after the stop codon of IL-21. Meanwhile, the genetic information of the major structures in this example is shown in Table 1 below.

[0065] [Table 1]

[0066]

[0067] Using the above structure, genetic structures of flgM-linker1-IL-21 (sequence number 6) and flgM-linker2-IL-21 (sequence number 7) were obtained through the gene synthesis service of Bionics Co., Ltd.

[0068] Subsequently, the above genetic construct was reacted with 10 U each of the enzymes NheI and SacI at 37°C for 2 hours. After electrophoresis of the reaction product, approximately 800 bp of DNA was identified using a Gel extraction kit (Qiagen Inc.) to obtain flgM-linker1-IL-21 insert and flgM-linker2-IL-21 insert.

[0069] Subsequently, to obtain flhDC insert DNA, PCR products containing the flhDC gene were obtained by PCR using the flhD-sac1-F and flhC-Sal1-r primers from Table 2, with the genomic DNA of wild-type Salmonella LT2 used as a template. The specific PCR conditions are as follows: 5 μl of 10x buffer, 10 μl of 5xQ solution, 7.5 μl of 2 mM dNTPs, 2.5 μl of 20 μM flhD-sac1-f primer, 2.5 μl of 20 μM flhC-Sal1-r primer, 1 μl of Taq polymerase, 2 μl of 10 ng / μl genomic DNA, and 19.5 μl of water were mixed, and PCR was performed using a Qiagen system under conditions of 95°C for 5 minutes, followed by 30 cycles of 94°C for 30 seconds, 49°C for 30 seconds, and 72°C for 1 minute, and then 72°C for 10 minutes. 1 μg of the PCR fragment purified with a PCR purification kit was reacted with 10 U each of sacI and SalI at 37°C for 2 hours, and the reaction product was purified with a PCR purification kit to construct flhDC insert DNA (Sequence No. 3).

[0070] [Table 2]

[0071]

[0072] 1-2. Preparation of Plasmids

[0073] Subsequently, using the pBAD18 asd+ plasmid as a vector, 10 U each of Nhe I and Sac I enzymes were reacted at 37°C for 2 hours, and the reaction products were purified using a PCR purification kit to obtain the respective vector DNAs. The asd gene inserted into the plasmid may consist of the nucleotide sequence of SEQ ID NO. 10. Subsequently, each vector DNA was ligated with the flgM-linker-IL-21 insert DNA at 25°C for 30 minutes and transformed into DH5a competent cells. Afterward, the transformed cells were plated on LB amp(ampicillin) solid medium and cultured at 37°C to select antibiotic-resistant colonies. Six candidate colonies were selected, reacted with 10 U each of Nhe I and Sac I at 37°C for 1 hour, and the formation of an approximately 800 bp band was confirmed by electrophoresis. In addition, the nucleotide sequence analysis of the corresponding candidate group was performed on the above plasmid to confirm the insertion of the flgM, linker, and IL-21 genes.

[0074] Subsequently, 1 μg of each of the above plasmids was reacted with 10U of sacⅠ and SalⅠ at 37°C for 2 hours, and the reaction product was purified using a PCR purification kit to complete the vector DNA. Afterward, each vector DNA was ligated with the flhDC insert DNA at 25°C for 30 minutes, and then DH5a competent cells were transformed. The transformed cells were plated on LB amp solid medium and cultured at 37°C to select antibiotic-resistant colonies. Six candidate colonies were selected and reacted with 10U of sacⅠ and SalⅠ at 37°C for 1 hour, and the formation of bands was confirmed by electrophoresis. Sequence analysis of the candidate colonies was performed to confirm the insertion of the flhDC gene.

[0075] Through this, flgM-linker1-IL-21-flhDC-pBAD18-asd+ and flgM-linker2-IL-21-flhDC-pBAD18-asd+ plasmids were prepared.

[0076] Example 2: Preparation of an attenuated Salmonella strain secreting flgM-IL-21

[0077] Attenuated Salmonella strains were transformed using the plasmid prepared in Example 1 above. Candidate attenuated Salmonella strains included BRD509 asd galE:tetRA-(#1317) and BRD509 asd rcsB galE:tetRA-(#1323). Strains were used and were obtained from Chungnam National University. The BRD509 Salmonella strain is a strain in which the functions of the aroA and aroD genes have been lost. The genes and strain information of the transformed strain are shown in Table 3 below.

[0078] [Table 3]

[0079]

[0080] Example 3: Confirmation of protein expression and secretion ability

[0081] To confirm the expression and secretion ability of flgM-IL-21 of attenuated Salmonella strains transformed with a recombinant expression vector according to one embodiment, the expression level of the protein was measured by Westot blot in the following manner.

[0082] A single colony of each of the above-mentioned transformed strains was inoculated into LB amp liquid medium and cultured with shaking at 37°C for 12-16 hours. Subsequently, the culture was diluted in fresh LB amp liquid medium to an OD600 of 0.05 and cultured with shaking at 37°C for 2 hours. When the OD600 reached 0.4-0.6 due to shaking, arabinose was added to a concentration of 0.2% (w / v), and the culture was cultured with shaking for an additional 5 hours at 37°C.

[0083] Afterward, 1 ml of whole cell culture (lanes 1 and 2) and another 1 ml of culture were centrifuged to separate the pellet (lanes 3 and 4) and the supernatant (lanes 5 and 6) to prepare the samples. The samples were divided into those not treated with arabinose (lanes 1, 3, and 5) and those treated (lanes 2, 4, and 6) at a concentration of 0.2% (w / v) (final). After culturing the strains, the culture medium was centrifuged at 8000 rpm to prepare each sample. The supernatant was then completely removed by 0.2 µm filtration and concentrated using nanosep (OD010C35 3K omega). 50 μl of each obtained sample was mixed with SDS sample buffer and denatured in a 100°C heating block for 5 minutes. Subsequently, the mixture was centrifuged at 13,000 rpm for 5 minutes at 4°C and subjected to electrophoresis on a 10% polyacrylamide gel. The gel was transferred using PVDF (polyvinylidene fluoride) to move proteins to a PVDF membrane, and the membrane was blocked using a blocking buffer at room temperature for 1 hour. Afterward, the primary antibody anti-his tag (SB194b, Southern Biotech) was applied, diluted 1 / 1000, and reacted at 4°C for 16 hours. Afterward, the membrane was washed three times at 10-minute intervals with TBST (Tris-buffered saline with 0.1% Tween-20), and then reacted with the mouse anti-mouse IgG (#7076s, Cell signaling, Danvers, MA, USA) secondary antibody at room temperature for 1 hour. After the reaction, it was washed three times at 10-minute intervals with TBST, exposed to X-ray film using ECL buffer, and then developed to check the expression levels of each protein.

[0084] In Figure 4, lanes 2, 4, and 6 are lanes treated with arabinose (final concentration 0.2% (w / v)), and lanes 1, 3, and 5 are lanes not treated with arabinose. Additionally, lanes 1 and 2 are results for the whole cell culture, 3 and 4 for the pellet, and lanes 5 and 6 for the supernatant.

[0085] As shown in Fig. 4, lanes 2, 4, and 6 treated with arabinose contain a larger amount of flgM- at the same concentration than lanes 1, 3, and 5 not treated with arabinose. IL-21 was expressed. In addition, the highest amount of flgM-IL-21 was found in lane 6 (supernatant) of strain #1323 transformed with a recombinant basal vector containing linker1.

[0086] Based on the above results, flgM-IL-21 expression is regulated by arabinose, and flgM-linker1-IL-21-flhDC-pBAD18-asd+ / BRD509 asd rcsB galE:tetRA-(#1323) It was confirmed that the strain exhibited the best extracellular protein secretion ability without bursting the cells.

[0087] Accordingly, the inventors of the present invention [obtained] the test group flgM-linker1-IL-21-flhDC-pBAD18-asd+ / BRD509 asd rcsB galE:tetRA-(#1323) The strain was deposited with the National Institute of Biological Resources, Korea Research Institute of Biotechnology and Bioengineering on July 14, 2023, accession number KCTC15503BP(S-flgM-mIL21-flhDC-pBAD18-ASD+ / #1323) was granted.

[0088] Example 4: Evaluation of anticancer effect using a mouse model

[0089] 4-1. Establishment of a mouse model xenografted with tumor cells

[0090] In this embodiment, the experimental animals were 6-week-old animals weighing approximately 20g. BALB / c female mice were purchased and used. According to the approved protocol, all animals Management, experimentation, and euthanasia were performed. Murine CT26 colon carcinoma cells were purchased from the American Cell Line Bank (ATCC) and cultured in DMEM medium containing 10% FBS and 1% penicillin-streptomycin. After collecting the CT26 tumor cells cultured in vitro, they were suspended in 20 μl PBS, and then 5×10 5 Tumor cells were xenografted by subcutaneously injecting cells into the right back of BALB / c mice. After approximately 2 weeks, the tumor size was 300 mm 3 When reached flgM-linker1-IL-21-flhDC-pBAD18-asd+ / BRD509 asd rcsB galE:tetRA-(#1323) Dilute the strain with PBS to approximately 1 × 10⁶ 7 It was injected via the tail vein at a dose of CFU / mice (0 day).

[0091] 4-2. Confirmation of Anticancer Effect

[0092] Subsequently, L-arabinose 80 mg / mice was injected intraperitoneally daily into the mice to induce the expression or secretion of flgM-IL-21, and the mouse body weight and tumor size were measured and recorded every two days. Tumor volume was calculated using the formula (tumor length × tumor width × tumor height) / 2, and in accordance with the guidelines of the Animal Ethics Committee, when the tumor volume was 1,500 mm² 3 When abnormalities occurred, the animal was euthanized. Fig. 5 is a schematic diagram showing the treatment process for a tumor mouse model of a transformed attenuated Salmonella strain according to one embodiment.

[0093] As shown in FIGS. 6 and 7, it was confirmed that the group in which the expression or secretion of IL-21 was induced by arabinose using the transformed attenuated Salmonella strain according to one embodiment significantly inhibited tumor growth. Furthermore, it was confirmed that while exhibiting excellent anticancer effects, there was no significant change in mouse body weight, indicating minimal side effects.

[0094] From this, it was found that the transformed attenuated Salmonella strain according to one embodiment is safe and has excellent anticancer effects, so it can be utilized as an active ingredient for the treatment of cancer.

[0095] Foregoing, specific parts of the present invention have been described in detail. It will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

[0096] Depository Name: Korea Research Institute of Biotechnology and Bioengineering Biological Resource Center (KCTC) Trustee Number: KCTC15503BP Date of Deposit: 2023-07-14

Claims

Claim 1 A recombinant expression vector comprising a flgM gene; an interleukin-21 gene; and a flhDC gene, wherein the flgM gene; the interleukin-21 gene; and the flhDC gene are operably linked to an inducible promoter, and the interleukin-21 gene is composed of the polynucleotide sequence of SEQ ID NO.

2. Claim 2 A recombinant expression vector according to claim 1, wherein the flgM gene; the interleukin-21 gene; and the flhDC gene are regulated for expression by the same inducible promoter. Claim 3 The recombinant expression vector of claim 1, wherein the recombinant expression vector comprises a flgM gene consisting of the polynucleotide sequence of SEQ ID NO. 1 and a flhDC gene consisting of the polynucleotide sequence of SEQ ID NO.

3. Claim 4 A recombinant expression vector according to claim 1, wherein the flgM gene and the interleukin-21 gene are connected through a linker. Claim 5 A recombinant expression vector according to claim 4, wherein the linker is composed of the polynucleotide sequence of SEQ ID NO.

4. Claim 6 Attenuated Salmonella strain transformed with the recombinant expression vector of claim 1. Claim 7 In claim 6, the attenuated Salmonella strain is an attenuated Salmonella strain in which the function of the genes asd, rcsB, and galE is lost. Claim 8 In claim 6, the attenuated Salmonella strain is an attenuated Salmonella strain having enhanced secretion ability for interleukin-21 protein. Claim 9 In claim 6, the attenuated Salmonella strain is the attenuated Salmonella strain deposited under accession number KCTC15503BP. Claim 10 A pharmaceutical composition for treating cancer comprising the attenuated Salmonella strain of claim 6 as an active ingredient, wherein the cancer is colon cancer or rectal cancer.

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